Project Summary The brain generates behavior through the concerted action of diverse cell types. Recent work has genetically identified cell types governing specific instinctive behaviors, such as neurons expressing galanin within the medial preoptic area (MPOA), which are are necessary for parenting, or Agrp and POMC-expressing neurons of the arcuate nucleus, which are critical for feeding. However, it is not well understood how and when cell types important to instinctive behaviors are defined during postnatal development. The periaqueductal gray (PAG) is thought to serve as an integrator of many forebrain signals. It receives major input from the hypothalamus, amygdala, and prefrontal cortex, and transmit proper signals to motor nuclei of the brainstem and spinal cord. While the PAG is necessary for specific functions present at birth, such as vocalization and suckling, other functions of the PAG, including mating and aggression are not manifested until later in life. This leads to the hypothesis that cell types necessary to control these behaviors are not mature until later in life. The goal of this proposal is to discover how and when cells gain their mature functional significance in the PAG. The findings from this proposal will provide insight about PAG-related behavioral pathologies and how they may arise, for example chronic anxiety and excessive aggression. Aim 1 will define PAG cell types and their postnatal developmental trajectories utilizing single nucleus RNA- sequencing from mice at P1, P7, P14, P28, and P50-P60 (adult). Aim 2 will spatially define the development and distribution of cell types in the PAG using Multiplexed Error Robust Fluorescent In Situ Hybridization. Aim 3 will determine PAG cell types active in age-dependent innate behaviors (P1/P7, vocalization; P14, fear; P28, aggression; adult, mating and parenting) using a panel of 20 immediate early genes as a readout of recent cellular activity. Preliminary data shows that cells increase the overall number of genes expressed with age. Using data computationally aligned across ages, preliminary developmental trajectories of several clusters show age- dependent increases in genes relevant for receptivity and signaling, such as neuropeptides and their receptors. This supports the hypothesis that with age, cells gain new functional capacities through upregulation of genes that allow for increased cellular crosstalk. This proposal seeks to define these functions and specifically identify which genes are upregulated concordantly with that function.